Synthesis of CuxO/Ag nanoparticles on exfoliated graphene: application for enhanced electrochemical detection of H2O2 in milk

In this paper, a novel composite is constructed as a non-enzymatic hydrogen peroxide (H2O2) sensor by liquid-phase exfoliation method, which is composed of copper oxide, cuprous oxide and silver nanoparticles doped few-layer-graphene (CuxO/Ag@FLG). Its surface morphology and composition were characterized by scanning electron microscopy (SEM) and X-ray photo spectroscopy (XPS), and its H2O2 sensing performances include catalytic reduction and quantitative detection were studied with electrochemical methods. Our sensor had a high sensitivity of 174.5 μA mM−1 cm−2 (R2 = 0.9978) in an extremely wide range of concentrations from 10 μM to 100 mM, a fast response (about 5 s) and a low limit of detection (S/N = 3) of 2.13 μM. The sensor exhibits outstanding selectivity in the presence of various biological interference, such as dopamine, ascorbic acid, uric acid, citric acid, etc. In addition, the constructed sensor continued 95% current responsiveness after 1 month of storage further points to its long-term stability. Last but not least, it has a good recovery rate (90.12–102.00%) in milk sold on the open market, indicating that it has broad application possibilities in the food industry and biological medicine.

H 2 O 2 is commonly used as a bleaching agent, disinfectant, and preservative in the food industry, owing to its strong oxidation resistance, good antibacterial and bactericidal properties 1 . H 2 O 2 is not only one of the byproducts of many classical enzymatic reactions, such as glucose oxidation, uric acid oxidation, oxalic acid oxidation, amino acid oxidation, glutamic acid, lysine oxidation, etc. 2 , but also plays a significant role as the primary messenger molecule in the cellular and redox metabolism 3 . It is worth noting that H 2 O 2 has strong oxidizability, ingesting residual H 2 O 2 in food will consume antioxidant substances in the body, accelerate the aging process of the human body, and reduce the resistance after ingestion. Excessive H 2 O 2 in the body will produce a large number of OH·, which participates electron transfer and hydroxylation reaction in biochemical process, damage cells, induce gene mutation and various diseases, even cause human cell canceration 4 . In 2017, the list of carcinogens was announced by International Agency for Research on Cancer of the World Health Organization, and H 2 O 2 appeared in the list of Class III carcinogens 5 . At present, it has been detected in the market that excessive amounts of H 2 O 2 residues exist in prepackaged milk, beverages, soy products, aquatic products, chicken feet, etc., posing a serious threat to human health. Therefore, the newly revised National Food Safety Standard in China (GB 2760-2014) stipulates that H 2 O 2 can be used as a food processing aid in various food processing processes, and the residual amount does not need to be limited, but it needs to be removed from the finished product. The Food and Drug Administration (FDA) requires H 2 O 2 to be used as an antimicrobial agent in milk, with a residual amount not exceeding 0.05 wt% in 21 CFR 184.1366. In addition, it is also stipulated that H 2 O 2 solution should be used in the sterilization of food packaging materials, and the residual amount of H 2 O 2 in distilled water after packaging should be less than 0.5 parts per million (testing should be conducted immediately after packaging) in 21 CFR 178.1005. Therefore, sensitive detection of H 2 O 2 is crucial for monitoring the quality of food and for applications of clinical, and biomedical. www.nature.com/scientificreports/ Nowadays, the methods used in the detection of H 2 O 2 include titration, spectroscopy, chromatography, chemiluminescence and electrochemical methods 6 . The Chinese national standard GB 5009. 226-2016 stipulates that the determination methods for H 2 O 2 in food residues are iodometry and titanium salt colorimetry. The quantitative limit of the above two methods is 3 mg kg −1 . These two methods are cumbersome, time-consuming, poor selectivity, and low accuracy, making it difficult to achieve trace detection. However, electrochemical method has the advantages of rapid response, rapid response, high-cost efficiency, simple operation, high selectivity and high sensitivity 7 . The first generation electrochemical H 2 O 2 detector relies on the reaction of enzyme with hydrogen peroxide. This enzyme-based technology has been mature and can now provide high sensitivity, high selectivity and low background noise 8 . Nevertheless, the complex process of immobilizing the enzyme on the electrode, strict conditions to maintain the enzyme activity, and the lack of long-term stability and reproducibility limit the application of this technology. More importantly, almost all enzymes are made of proteins, which will hinder the electrochemical reaction on the surface of electrode 9 . Therefore, non-enzymatic electrochemical H 2 O 2 detection have been developed to eliminate these shortcomings due to its faster and more effective electron transport 10 .
Enzyme-free H 2 O 2 sensors generally prepared by precious metal nanoparticles (NPs) include Au, Ag, Pt and Pd NPs 11 , As a precious metal, Ag NPs have large specific surface area, good stability, good biocompatibility, excellent conductivity and electrocatalytic activity 12 16 . CuO and Cu 2 O have also been developed to prepare H 2 O 2 sensors, which are inexpensive and strong stability, but with low sensitivity and narrow linear detection range. However, metal NPs have limited catalytic ability, because they are easily oxidized, and aggregative owing to the existence of van der Waals force between NPs 17 . In order to improve this problem, a suitable carrier is needed. At present, the substrates that have been widely studied are usually carbon materials, including ordered mesoporous carbon, carbon nanotubes, carbon nanofibers and graphene 18 . Among them, graphene has high specific surface area, good thermal stability, good conductivity, stability and high electron transfer ability, and its surface defects can anchor metal NPs, so it is an excellent carrier material 19 . Most of the existing related studies use graphene oxide prepared by Hummer's method as carbon substrate to prepare metal nanoparticle composites. The preparation process is complex, and the distribution position and size of metal particles are uneven, which affects the sensing performance of the composites 20 . The liquid phase exfoliated method is low-cost, simple and controllable, green and environmentally friendly, and easy to large-scale production. It has been studied for use in Cd 2+ and Pb 2+ sensors, as well as flavomycin sensors 20,21 .
In this work, we have used water-in-oil emulsion as a stabilizing system to synthesis Cu x O/Ag@FLG composite by a simple ultrasonic exfoliation method for the first time. After a series of characterization, the morphology and structure of the composite were studied and modified on a glassy carbon electrode (GCE) to explore the detection of H 2 O 2 performance. Compared with the reported Ag, CuO or Cu 2 O NPs for H 2 O 2 sensors, the Cu x O/ Ag@FLG/GCE H 2 O 2 electrochemical sensor has the advantages of simple preparation, low cost and stable physicochemical properties. Our electrochemical experiments show the prepared sensor displays wide linear range, high sensitivity and selectivity toward the reduction of H 2 O 2 with a low detection, and we also demonstrate it is successfully used to detect the concentration of H 2 O 2 in milk. This study would offer a new routine for developing graphene-based electrochemical sensors for detecting H 2 O 2 . Synthesis of Cu x O/Ag@FLG composite. 0.4 g graphite powder, 5 mL 3 wt% CuSO 4 in 2 wt% PVP solution, 3 g Tween 80, 4 g Span 80, 5 mL anhydrous ethanol are added into 30 mL cyclohexane, stable emulsion can be formed through vortex mixing. Then, ultrasonic unit is used to exfoliate the above solution for 2 h, with 40 kHz ultrasonic power. After that, 0.057 g NaBH 4 is added into the emulsion above and exfoliate the above solution for 2 h. Finally, the solid were carefully collected by filter to obtain Cu x O@FLG. 0.4 g Cu x O@FLG, 5 ml 3 wt% AgNO 3 in 2 wt% PVP and 20% NH 3 ·H 2 O mixing solution, 3 g Tween 80, 4 g Span 80, 5 mL anhydrous ethanol are added into 30 mL cyclohexane, stable emulsion can be formed through vortex mixing. Then, ultrasonic cleaner is used to exfoliate the above solution for 2 h. At the end, the solid were carefully collected by filter to obtain Cu x O/Ag@FLG. www.nature.com/scientificreports/ GCE and dried under an infrared drying lamp for 10 min to prepare Cu x O/Ag@FLG/GCE. For comparison, we prepared Cu x O@FLG/GCE and Ag@FLG/GCE using the same method.

Fabrication of Cu
Analytical procedure. JSM-7610F PLUS scanning electron microscope (SEM, JEOL, Tokyo, Japan), H-7650 transmission electron microscope (TEM, Hitachi, Tokyo, Japan), D/max-2200/PC X-ray diffractometer (XRD, Hitachi, Tokyo, Japan) and ESCALAB 250 X-ray Photoelectron Spectroscopy (XPS, Thermo Fisher Scientific, Massachusetts, America) were used to characterize the surface morphology and structure. Cyclic Voltammetry (CV), electrochemical impedance spectroscopy (EIS) and Chronoamperometry (i-t) were determined by the CHI660E electrochemical workstation (CH Instruments, Shanghai, China). Electrolyte was consisting of phosphoric acid buffer solution, the working electrode was modified GCE, the counter electrode was platinum sheet, the reference electrode was saturated calomel electrode (SCE). The i-t test was performed by adding H 2 O 2 within a time interval of 40 s.

Results and discussion
Characterizations of morphology. The construction and detection principles of the Cu x O/Ag@FLG/ GCE sensor are summarized in Fig. 1a. Next, the morphology of the material was characterized. The SEM image depicted in Fig. 1b shown that FLG with distinct crumples surface and large size were obtained, and there are lots of Ag nanoparticles and flower-like Cu x O nanosheet supported on the surface of crumble layers of exfoliated graphene. Furthermore, EDS images shown in Fig. 1c confirm the existence and homogenous distribution of C, O, Cu, Ag elements. Two-dimensional conductive network structure has been developed in Cu x O/Ag@FLG, which is conducive to the rapid transfer and conduction of electrons, so as to detect target analytes sensitively. As a supplement, the micromorphologies and microstructures of synthetic Cu x O/Ag@FLG investigated by TEM. As shown in Fig. 2a,b, the metal NPs were successfully loaded on the graphene sheet. The lattice spacing in  (Fig. 2d,e). In addition, the Raman spectrum compares the graphite powder raw material with the prepared material, as shown in Fig. 2f and Table 1. The peak position of the G peak and 2D peak move to the low frequency direction, and the half peak width of 2D peak decreases, and the I 2D /I G increases, which indicate the successful preparation of FLG 22 . Besides, the same trend as TEM images could be observed in XRD pattern. As can be seen from Fig. 2g, the strong peak at 26.38°  Compared to graphene oxide, the above C 1s XPS spectrum is more similar to reduced graphene oxide, because the peak intensity for C-C are significantly stronger than C-O and C=O 23 . Figure 3c indicated that the characteristic peaks at 531.6 eV, 532.5 eV which belong to lattice oxygen of Cu 2 O and CuO 24 , respectively, and the peaks at 533.5 eV, 530.3 eV are usually attributed to O in adsorbed -OH groups or carbonates 25 . The Cu 2p spectrum (Fig. 3d) (Fig. 3e) was deconvoluted into two peaks, the peak of Ag 3d 5/2 at 368.6 eV and the peak of Ag 3d 1/2 at 374.6 eV are attributed to Ag 028 . Fig. 4, the interfacial characteristics of the modified electrodes were investigated by EIS. Generally, the diameter semicircle at high frequency represents the resistance to charge transfer (Rct) 29 . In Fig. 4a, compared with the Rct of bare GCE (5774 Ω), the Rct increases to 12,154 Ω after modified with Cu x O@FLG by reason of weak conductivity of Cu 2 O and CuO. However, the Rct decreases to 2464 Ω when modified with Ag@FLG, which indicates that Ag@FLG can promote electron transfer greatly. Furthermore, It is worth noting that Cu x O/Ag@FLG/GCE has the smallest Rct (1605 Ω), which illustrates its excellent electron transport ability owing to the large surface area and the potential synergism of the nanocomposites.

Electrochemical detection of H 2 O 2 . Electrochemical reduction of H 2 O 2 by the synthesized composite
was studied using CV method. Therefore, the CV curves are shown in Fig. 5 in N 2 -saturated 0.01 M PBS contained 4 mM H 2 O 2 at a scan rate of 50 mV s -1 . The reduction peak current enhancement of Cu x O@FLG/GCE is extremely weak (Fig. 5a). In addition, Ag@FLG show higher current response thanks to exceptional electrocatalytic activity of Ag NPs. CV curve of Cu x O/Ag@FLG/GCE has shown a lower reduction potential at − 0.65 V and higher current of 80 μA. Therefore, the Cu x O/Ag@FLG/GCE is a prospective electrochemical sensor for H 2 O 2 detection. With the increase of H 2 O 2 addition, the cathodic current increases linearly in Fig. 5b. Subsequently, the CV curves of Cu x O/Ag@FLG at varying scan rates (10-120 mV s −1 ) in 0.01 M PBS (pH 7.4) contained 4 mM H 2 O 2 were investigated (Fig. 5c). Along with the square root of the scanning speed, the reduction peak current increases linearly, and correlation coefficients (R 2 ) was fitted as 0.9803, which indicates that the diffusion-controlled electrochemical process (Fig. 5d,e)  were optimized which include volume of catalyst ink, pH of supporting electrolyte and applied potential. The response current is the largest when the volume of ink was 5 µL (Fig. 6a). Furthermore, volume of 5 µL Cu x O/ Ag@FLG composite on GCE is used for further studies. As shown in the Fig. 6b, CV curves was tested in PBS with different pH containing 4 mM H 2 O 2 , as a result, the maximum current response appears in PBS with pH www.nature.com/scientificreports/ 7.4. Therefore, PBS with pH 7.4 was selected as the electrolyte solution. The influence of different applied potentials on sensitivity was investigated by i-t method (Fig. 6c). The sensitivity was the highest at − 0.65 V. Hence, − 0.65 V is selected as the detection potential in the following studies. Under the best conditions (5 μL Cu x O/Ag@FLG ink, 0.01 PBS with pH 7.4, − 0.65 V), i-t curve was plotted for Cu x O/Ag@FLG/GCE as H 2 O 2 was added every 40 s (Fig. 7a). The response time of Cu x O/Ag@FLG/GCE to H 2 O 2 is about 5 s. Meanwhile, the linear relationship between concentration of H 2 O 2 and current is fitted (Fig. 7b)  After adding interferent, the current response to H 2 O 2 on Cu x O/Ag@FLG/GCE at the detection potential is very small, which indicates that the response of the sensor to H 2 O 2 is almost not affected in the presence of other compounds, Cu x O/Ag@FLG/GCE shows high selectivity. Long-term stability and reproducibility are momentous factors which evaluate the performance of sensors. Five Cu x O/Ag@FLG/GCE electrodes were prepared respectively, and the relative standard deviation (RSD) of current response in the same concentration of H 2 O 2 was 3.69%, which presents a good repeatability (Fig. 8b). After storing at room temperature for 1 month, the reaction of the prepared electrode to H 2 O 2 decreased to about 95% (Fig. 8c)   www.nature.com/scientificreports/

Conclusions
To summaries, Cu x O/Ag@FLG sensor has been prepared successfully by ultrasonic exfoliation method. Cu x O/ Ag@FLG/GCE electrode has shown excellent catalytic reduction performance for H 2 O 2 , such as wide linearity range (10-100, 000 μM), low detection limit (2.13 μM), high sensitivity (174.5 μA mM −1 cm −2 ) and long-term stability. In addition, the prepared sensor can detect H 2 O 2 in milk samples, which makes it possible for quality control in food industry, suggesting that as-synthetic Cu x O/Ag@FLG has a broad application prospect in electrochemical sensors.  www.nature.com/scientificreports/

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.